基孔肯雅病毒E2蛋白的原核表达及纯化Prokaryotic expression and purification of E2 protein of Chikungunya virus
郭丹丹;曹亮;田明尧;鲁会军;孙文超;张涵;汪伟;刘云霞;金宁一;郭焱;
摘要(Abstract)：
目的原核表达并纯化基孔肯雅病毒(Chikungunya virus,CHIKV)E2主要结构蛋白,为预防CHIKV感染及亚单位疫苗的研究奠定基础。方法根据Gen Bank上公布的CHIKV基因序列合成E2基因,并进行密码子优化,以提高原核表达效率。设计引物并以合成的E2基因为模板,PCR扩增CHIKV E2,连接到原核表达载体p ET-28a上,构建重组原核表达质粒p ET-28a-CHIKV E2,转化大肠埃希菌BL21(DE3),IPTG诱导表达。表达产物经SDS-PAGE及Western blot鉴定后进行纯化。结果重组原核表达质粒p ET-28a-CHIKV E2经双酶切鉴定构建正确,测序结果与原序列一致。表达的CHIKV E2融合蛋白相对分子质量约为47 000,可与His标签抗体特异性结合,纯化后纯度达95%以上。结论成功在大肠埃希菌中表达了CHIKV E2蛋白,纯化后蛋白纯度较高,为CHIKV亚单位疫苗的免疫试验奠定了基础。
关键词(KeyWords)： 基孔肯雅病毒;原核表达;纯化
基金项目(Foundation): 国家重点研发计划项目(重要新发突发病原体防治、处置技术与产品研究,2016YFC1200900)
作者(Authors): 郭丹丹;曹亮;田明尧;鲁会军;孙文超;张涵;汪伟;刘云霞;金宁一;郭焱;
DOI: 10.13200/j.cnki.cjb.002273
参考文献(References)：
- [1]BURT F J,ROLPH M S,RULLI N E,et al.Chikungunya:a re-emerging virus[J].Lancet,2012,379(9816):662-671.
- [2]WEAVER S C,LECUIT M.Chikungunya virus infections[J].N Engl J Med,2015,373(1):94-95.
- [3]SERGON K,NJUGUNA C,KALANI R,et al.Seroprevalence of Chikungunya virus(CHIKV)infection on Lamu Island,Kenya,October 2004[J].Am J Trop Med Hyg,2008,78(2):333-337.
- [4]PIALOUX G,GA譈Z魪RE B A,JAUR魪GUIBERRY S,et al.Chikungunya,an epidemic arbovirosis[J].Lancet Infect Dis,2007,7(5):319-327.
- [5]LIN M,LI H,HUANG J C,et al.First imported Chikungunya fever case at Guangzhou Baiyun international port[J].Chin Front Health Quarant,2008,31(4):221-223,236.(in Chinese)林苗,李华,黄吉城,等.广东检验检疫局发现中国内地首例输入性基孔肯雅热病例[J].中国国境卫生检疫杂志,2008,31(4):221-223,236.
- [6]WANG D H,DONG Z Q,CHEN C,et al.Investigation and analysis of the first case of imported Chikungunya fever in Guangzhou[J].Prog in Microbiol Immunol,2010,38(2):54-56.(in Chinese)王大虎,董智强,陈纯,等.广州首起输入性基孔肯雅热的调查分析[J].微生物学免疫学进展,2010,38(2):54-56.
- [7]SUN Q X,SHI L,SUN K J,et al.Chikungunya virus seroepidemiological survey on international travelers at the port between Shenzhen and Hongkong[J].Mod Prev Med,2014,41(8):1348-1350.(in Chinese)孙秋香,史蕾,孙康健,等.深港口岸入境旅客基孔肯雅病毒血清流行病学调查[J].现代预防医学,2014,41(8):1348-1350.
- [8]WEAVER S C,LECUIT M.Chikungunya virus and the global spread of a mosquito-borne disease[J].N Engl J Med,2015,372(13):1231-1239.
- [9]WEAVER S C,FORRESTER N L.Chikungunya:evolutionary history and recent epidemic spread[J].Antiviral Res,2015,120(1):32-39.
- [10]SCHWARTZ O,ALBERT M L.Biology and pathogenesis of chi-kungunya virus[J].Nat Rev Microbiol,2010,8(7):491-500.
- [11]SNYDER J E,KULCSAR K A,SCHULTZ K L,et al.Functional characterization of the alphavirus TF protein[J].J Virol,2013,87(15):8511-8523.
- [12]AHOLA T,COUDERC T,NG L F,et al.Therapeutics and vaccines against chikungunya virus[J].Vector Borne Zoonotic Dis,2015,15(4):250-257.
- [13]SARASWAT S,ATHMARAM T N,PARIDA M,et al.Expression and characterization of yeast derived Chikungunya virus like particles(CHIK-VLPs)and its evaluation as a potential vaccine candidate[J].PLo S Negl Trop Dis,2016,10(7):e0004782.doi:10.1371/journal.pntd.0004782.
- [14]VAN BORTEL W,DORLEANS F,ROSINE J,et al.Chikungunya outbreak in the Caribbean region,December 2013 to March 2014,and the significance for Europe[J].Euro Surveill,2014,19(13).pii:20759.
- [15]MCCLAIN D J,PITTAMAN P R,RAMSBURG H H,et al.Immunologic interference from sequential administration of live attenuated alphavirus vaccines[J].J Infect Dis,1998,177(3):634-641.
- [16]HOKE C H Jr,PACE-TEMPLETON J,PITTMAN P,et al.US Military contributions to the global response to pandemic chikungunya[J].Vaccine,2012,30(47):6713-6720.
- [17]KHAN M,DHANWANI R,RAO P V,et al.Subunit vaccine formulations based on recombinant envelope proteins of Chikungunya virus elicit balanced Th1/Th2 response and virusneutralizing antibodies in mice[J].Virus Res,2012,167(2):236-246.
- [18]AKAHATA W,YANG Z Y,ANDERSEN H,et al.A viruslike particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection[J].Nat Med,2010,16(3):334-338.
- [19]METZ S W,GARDNER J,GEERTSEMA C,et al.Effective chikungunya virus-like particle vaccine produced in insect cells[J].PLo S Negl Trop Dis,2013,7(3):e2124.
- [20]RUEDA P,FOMINAYA J,LANGEVELD J P,et al.Effect of different baculovirus inactivation procedures on the integrity and immunogenicity of porcine parvovirus-like particles[J].Vaccine,2000,19(7-8):726-734.
- [21]NUC P,NUC K.Recombinant protein production in Escherichia coli[J].Postepy Biochem,2006,52(4):448-456.